To investigate the regulatory effects of the nucleus accumbens (NAcSh)-lateral hypothalamus (LHA) GABAergic neural pathway on palatable food (PF) intake via orexin-A expression in diet-induced obesity (DIO) rats. NAcSh-LHA GABAergic pathways were observed by fluorogold retrograde tracing combined with fluorescence immunohistochemistry, and the regulatory effects of this neural pathway on PF intake were detected after 1) microinjection of GABA-A receptor agonist muscimol (MUS) or antagonist bicuculine (BIC) into LHA, 2) electrical stimulation NAcSh, and 3) blocking the orexin-A receptor by icv SB334867. P<0.05). Microinjection of MUS into LHA significantly decreased the PF intake in both ND and DIO rats (P<0.05), and BIC could markedly increase the PF intake in the ND rats (P<0.05), but not the DIO rats (P>0.05). After NAcSh electrical stimulation or SB334867 ICV injection, the PF intake was significantly decreased in the DIO rats (P<0.05), and there was no significant difference after preadministration of BIC into LHA (P>0.05). Compared with rats on a normal diet (ND), NAcSh-LHA GABAergic neurons in the DIO rats were significantly decreased, and orexin-A expression in LHA significantly increased (This GABAergic pathway could regulate the expression of orexin-A in LHA and PF intake. Orexin-A neurons in LHA of DIO rats might be less sensitive to GABAergic signals and may consequently lead to more hedonic food intake. According to epidemiological investigations, the overweight condition and obesity have increased rapidly over the past 50 years, which greatly increases the risk of many diseases such as type 2 diabetes, hypertension, myocardial ischaemia, and so on . Obese p“Hedonic feeding” is believed to be regulated by reward-related brain regions, especially the shell of the nucleus accumbens (NAcSh) , 4. ThisOrexin-A is a type of orexinergic neuropeptide expressed in LHA, and the number of orexin-A immuno-positive neurons of LHA increases in high-fat diets . MoreoveTo verify this assumption, the endogenous orexin-A expressions in LHA of the diet-induced obesity (DIO)rats after PF intake were observed by immunofluro-chemistry. Then, the adaptive changes of the GABAergic neural pathway from NAcSh to LHA in the DIO rats were explored. Furthermore, the PF intake was recorded after administration of the agonist or the antagonist of the GABA-A receptor in LHA, after electrical stimulation of the NAc and after administration of orexin-A receptor antagonist SB334867 in the lateral ventricles to test the functions of this neural pathway. This study evaluated the roles of NAcSh-LHA GABAergic neural pathway, with a focus on downstream orexin-A signaling, to provide a potential novel treatment strategy for obesity.AnimalsIn all, 192 male Wistar rats were included in the study  20190003). The experimental animals were fed at a temperature of 22 ± 2 °C, a relative humidity of 58 ± 5%, and a light/dark cycle of 12 hr (8:00 am‒8:00 pm). Protocols were approved by the Qingdao University Animal Care and Use Committee.Experimental instruments and reagentsThe stereotaxic instrument was obtained from Scientifica (UK). Rabbit anti-GABA primary antibody, sheep anti-orexin-A primary antibody, rabbit anti-nesfatin-1 primary antibody, rabbit anti-GABA receptor and primary antibody were purchased from Abcam (UK). Sheep anti-rabbit Cy3 secondary antibody, donkey anti-rabbit Cy3 secondary antibody, and donkey anti-sheep FITC secondary antibody were purchased from Jackson ImmunoResearch (USA). GABA-A receptor agonist muscimol (MUS), GABA-A receptor antagonist bicuculline (BIC) and orexin-A receptor antagonist SB334867 were purchased from Sigma (USA). High-fat diet  was obtained from Qingdao Haizhida Biotechnology Co. (China). Sweetened condensed milk  was purchased from Nestle .Experimental methodsExperimental designExperiment 1: Twenty Rats were randomly divided into two groups: One group was given a normal diet (ND), and the other group was given a high-fat diet. Body weight was measured weekly. Eight weeks later, 6 ND rats and 6 DIO rats were randomly selected to observe their PF intake. Experiment 2: Six ND rats and 6 DIO rats were randomly selected to observe expression of orexin-A and nesfatin-1 in LHA after PF intake by fluorescence immunohistochemistry.Experiment 3: Six ND rats and 6 DIO rats were randomly selected to observe the coexistence of fluorogold and GABA immunoreactive neurons in the NAc and the co-expressions of orexin-A and GABA-A receptors in LHA by retrograde tracing combined with immunofluorescence histochemical staining. Experiment 4: Twenty-four ND rats and 24 DIO rats were randomly divided into three groups. The PF intake was monitored after normal saline (NS), GABA-A receptor agonist MUS, or GABA-A receptor antagonist BIC were microinjected into LHA in ND rats and DIO rats, respectively. Experiment 5: Thirty-two ND rats and 32 DIO rats were randomly selected and divided into four groups: (a) NS + sham stimulation group (SS); (b) NS + electrical stimulation group (ES); (c) BIC + SS; (d) BIC + ES. The palatable food intake in the ND and DIO rats was observed after the GABA-A receptor blocker BIC was injected into LHA, and NAc was stimulated by electrical stimulation.Experiment 6: Twenty-four ND rats and 24 DIO rats were randomly divided into three groups, (1) NS + NS, (2) SB334867 (SB) + NS, and (3) SB + BIC, to observe the effect of SB334847 i.c.v injection, an orexin-A receptor antagonist, on palatable food intake before administration of BIC in LHA of the ND and DIO rats.Diet-induced obesity rat modelad libitum.After 7 days of adaptive feeding, the rats were fed a high-fat diet, and their body weight was measured every week. After 8 weeks, the rats whose body mass exceeded 20% of the ND rats were defined as DIO rats. The remaining rats were fed normal chow pellets, and all rats drank tap water Palatable food intake nd, 4th, 6th, and 8th days to determine the SCM intake of rats.Before the experiment, the rats were raised alone, feeding and drinking freely. At 9:00 pm on the day of the experiment, SCM was provided to rats in the ND group and DIO group, respectively; the intake of SCM was recorded for 30 min/d. The method for determining the palatable food intake included weighing the SCM before and after the experiment and calculating the consumption on the 2Fluorescence immunohistochemistry2. The percentage of double-labelled cells (%) = the number of double-labelled cells/the total number of positive neurons × 100%.Rats were anaesthetised by thiobarbitol  and were perfused and fixed with 4% paraformaldehyde solution. The brains were dehydrated in 30% sucrose and then sliced into 20 μm thick coronal sections. Brain sections were blocked with sheep or donkey serum at room temperature for 2 hr. The sections were incubated overnight at 4 °C with primary antibody (sheep anti-orexin-A 1: 400/rabbit anti-nesfatin-1 antibody 1: 200/rabbit anti-GABA-A receptor 1: 300 /rabbit anti-GABA-A antibody 1:500). Later, the sections were washed three times with PBS and incubated with fluorescent-labelled secondary antibody (Cy3 1:300/FITC1:50) at room temperature for 2 hr, then washed again with PBS three times. Finally, the slices were sealed with anti-quenching sealant and observed with a fluorescence microscope. Using an image analysis system , five fields of five brain slices of LHA or NAc of each rat were counted for immunoreactive cells. The area of cell count was 350 × 350 μmFluorogold retrograde tracing4 U) was provided for 3 days to prevent infection. Seven days later, the rats were perfused with 4% paraformaldehyde solution. Then the brains were removed, and frozen sections were made. The sections were stained by fluorescence immunohistochemistry and the GABAergic projections from NAc to LHA were observed by a fluorescence microscope.After rats were anaesthetised by thiobarbitol , they were fixed on the stereotaxic instrument. According to the stereotaxic atalas of Paxinos and Watson , 0.2 μl Drug microinjection4 U penicillin for 3 days to prevent infection, and the follow-up experiment was conducted 1 week after the operation.The rats were fasted overnight and anaesthetised with thiobarbitol . Then, they were placed in the stereotaxic frame. A longitudinal incision was made, and the skull was fully exposed. According to the rat brain atlas , a 24 G Electrical stimulation of the NAcA monopolar stimulation electrode was inserted into the NAc  and connected with the stimulator. The stimulation parameters were as follows: 20 μA intensity, 0.5 ms duration, which lasted 1 hr at 50 Hz . The conHistological verificationAt the end of the experiment, each rat was perfused and fixed, and 50 µm frozen sections of the brain were selected to verify the locations of nuclear injection or electrical stimulation. Incorrectly positioned data were excluded from statistical analysis.Statistical processingpost hoc Bonferroni test were used for comparisons among means. P<0.05 was considered statistically significant.SPSS 17.0 statistical software was used to analyse the data. The experimental results were presented as mean ± SD. Comparisons were made between groups using the Student’s t-test (two groups only), and one-way or two-way ANOVA with The palatable food intake in DIO and ND ratsP<0.05). It indicated that the DIO rat model was established. Within 8 days after administration of PF, the intake of PF in the ND group and DIO group showed an increasing trend. The intake of PF in the DIO group on the 6th and 8th days were significantly higher than in the ND group  . These results indicated that some GABA neurons in NAc were projected to LHA, and the number of GABA neurons in the DIO rats markedly decreased.To observe whether theF changes in the orexin-A expression in LHA of DIO rats were due to the activation of the reward closely related nuclei NAc, a retrograde tracer FG was injected into LHA . Seven dThe effect of microinjection of MUS or BIC into LHA on PF intake in DIO ratsP<0.05, P<0.001, P<0.05, P>0.05, To explore the effect of the GABAergic pathway on the PF intake in the DIO rats, the GABA-A receptor agonist MUS or the antagonist BIC was bilaterally injected into LHA . AdminisEffect of NAcSh electrical stimulation on PF intake in DIO ratsP<0.05, P<0.05), suggesting that BIC could partially block this inhibitory effect of ES, as shown in P<0.05, P>0.05, Electrical stimulation of NAcSh was used to observe the effect of the GABAergic signal in NAcSh on the PF intake in the DIO rats . After eEffect of orexin-A signals blocking on the PF intake in the DIO ratsP<0.05, P>0.05, To verify whether orexin-A is the downstream signal of GABA to regulate the PF intake in rats, the effect of ICV injection of orexin-A receptor blocker SB334867 on PF intake was observed . The intIn this study, it was found that DIO rats had higher orexin-A expression in LHA and lesser GABAergic projection from NAcSh to LHA after PF intake. The change in the GABAergic pathway in the DIO rats influenced PF intake, and the orexin-A receptor antagonist could significantly decrease the PF intake in DIO rats.LHA is a key nucleus in the central regulation of food intake, which affects feeding behavior by secreting different neurotransmitters . Orexin-NAc is an important part of the mesencephalic limbic system and plays a key role in the reward of drug abuse and the regulation of natural rewards, such as food and sexual behavior , 18. NAcWe observed the NAc-LHA GABAergic pathway using fluorogold retrograde tracing. The results showed that FG and GABA coexisted in some neurons of NAcSh, which proved that GABA neurons in NAcSh could project to LHA. In addition, the number of GABA and FG double-labelled neurons in the NAc of the DIO rats was significantly less than that in the ND rats, indicating that the number of GABAergic neurons in the NAc projecting LHA decreased in the DIO rats. Next, the effects of microinjection of GABA-A receptor agonist MUS or antagonist BIC into LHA were explored, and the results suggested that the PF intake decreased significantly in the DIO rats when MUS was administrated, while it did not change as in the ND rats when BIC was used. It might be related to the decrease of GABAergic inhibitory signals in LHA of the DIO rats. The negative regulation of downstream neurons was weakened, and this might lead to the feeding signal orexin-A expression maintaining high expression levels. The orexin-A and GABA-A receptor coexist in some neurons of LHA in the ND rats and DIO rats, which suggested that the NAc-LHA GABAergic pathway might be involved in the regulation of orexin-A expression in LHA. Paul J. Kenny found that LHA damage could attenuate changes in food intake induced by stimulation of NAc, and the decrease in NAc activity improved the activity of LHA neurons , which wFurthermore, NAcSh electrical stimulation could affect the orexinergic peptide in LHA through the GABAergic signal pathway to suppress PF intake in the ND rats, while the inhibitory effect of GABA was reduced in the DIO rats.Based on the sustained high levels of orexin-A in LHA of the DIO rats, i.c.v. injection of SB334867 significantly decreased PF intake, and regardless of whether GABA-A receptor blockers were preadministrated in LHA, there was no significant effect on PF intake in rats. The adaptive changes of NAcSh to PF in the DIO rats can promote the intake of PF and increase energy intake by reducing the inhibitory effects on orexin-A expression in LHA.Our results demonstrated that GABA neurons in NAcSh participated in inhibitory effects on LHA. The signals of GABAergic neurons from the NAc to LHA decreased in obesity, resulting in a relative increase of orexigenic peptide in LHA and promoting the PF intake. The current study helped clarify the neural pathway from GABA neurons in NAcSh to the orexin-A neurons in LHA and also enriched the experimental basis of “hedonic feeding” and obesity.SG, FG study conception and design; JK, XH, NZ, JH, PJ, SY, XR, YW data processing, collection, perform experiment; SG, FG analysis and interpretation of results; JK, SG draft manuscript preparation, visualization; YG, FG critical revision or editing of the article; SG, FG Final approval of the version to be published; SG, FG supervision, funding acquisition. JK and SG were considered co-first authors.The authors declare that no conflict of interest exists.


Many neurodegenerative disorders, including Alzheimer’s disease (AD), are strongly associated with the accumulation of oxidative damage. Transgenic animal models are commonly used to elucidate the pathogenic mechanism of AD. Beta amyloid (Aβ) and tau hyperphosphorylation are very famous hallmarks of AD and well-studied, but the relationship between mitochondrial dysfunction and the onset and progression of AD requires further elucidation. In this study we used transgenic mice (the strain name is 5xFAD) at three different ages  as an AD model. Cognitive impairment in AD mice occurred in an age-dependent manner. Aβ1-40 expression significantly increased in an age-dependent manner in all brain regions with or without AD, and Aβ1-42 expression in the hippocampus increased at a young age. In a Western blot analysis using isolated mitochondria from three brain regions , NMNAT-3 expression in the hippocampi of aged AD mice was significantly lower than that of young AD mice. SOD-2 expression in the hippocampi of AD mice was lower than for the age-matched controls. However, 3-NT expression in the hippocampi of AD mice was higher than for the age-matched controls. NQO-1 expression in the cerebral cortex of AD mice was higher than for the age-matched controls at every age that we examined. However, hippocampal NQO-1 expression in 6-month-old AD mice was significantly lower than in 3-month-old AD mice. These results indicate that oxidative stress in the hippocampi of AD mice is high compared to other brain regions and may induce mitochondrial dysfunction via oxidative damage. Protection of mitochondria from oxidative damage may be important to maintain cognitive function. Alzheimer’s disease (AD) is a severe irreversible brain disorder. The number of patients suffering from it in Japan is currently higher than 3 million, and the rate of its increase there is the fastest in the world . The mosTo elucidate the mechanisms of AD onset and progression, researchers have used many experimental models, such as fruit flies , CaenorhReactive oxygen species (ROS) such as superoxide anion radicals are mainly produced in the mitochondrial electron transport chain ,13. SupeTo prevent AD onset and progression, identification of early changes in the brain is necessary, and many researchers have searched for suitable markers. In the early stage of AD, axonal degenerative effects, such as shrinkage and beading, appear in the brain ,19. ThesAll animal experiments were approved by the Animal Protection and Ethics Committee of Shibaura Institute of Technology , and 5xFAD transgenic mice  6799Vas/Mmjax, Alias/5XFAD) from The Jackson Laboratory  and self-bred prior to use in these experiments. In the present study, 3-, 6- and 20-month-old AD-transgenic mice were used. C57BL/6 Ncr male mice of the same age were obtained from Sankyo Labo Service Corp. Inc.  and used as a control group. All mice were maintained in conditions of controlled temperature (22 ± 2 °C) and a 12 h light/dark cycle, and were provided with free access to food and water. Food consisted of normal diet pellets (Labo MR Stock) purchased from Nosan Corp. . Cognition and motor function were assessed using tests as described below. Following assessments, the mice were euthanized, and brain samples , cerebellum (Cer), and hippocampus (Hip)) were collected for analysis. All other chemical agents were obtained from FUJIFILM Wako Pure Chemical Corp. .Cognitive function was assessed using a Morris water maze apparatus ,25. The The rota-rod  test was used to assess coordinated movement ability, as described previously with some modifications . The rodg for 10 min at 4 °C. The supernatant was centrifuged at 10,000× g for 10 min at 4 °C. The precipitate was diluted in lysis buffer and mixed with 5× sodium dodecyl sulfate (SDS) buffer.Mitochondria were isolated from the cerebral cortex, cerebellum and hippocampus of each mouse using a self-made buffer -1-piperazinyl] ethanesulfonic acid (HEPES), 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.2). After sonication, the samples were centrifuged at 500× All samples except isolated mitochondria samples were homogenized in lysis buffer and used in Western blotting as described previously , with sop values of less than 0.05 were considered statistically significant. The detailed statistical methods are described in the individual figure captions.Data are expressed as means ± standard error (SE). They were analyzed using GraphPad Prism 9.2.0 ; The results of the Morris water maze test are shown in The coordination abilities were measured using a rota-rod apparatus . The timWe measured Aβ protein expression using Western blotting, and measured band intensities for Aβ1-40 expression A. Aβ1-40We used 5xFAD mice in this experiment, in which no mutation at the site of tau phosphorylation is present. To analyze the relationship between Aβ accumulation and the ratio of tau protein including the phosphorylated form in AD mice, we analyzed tau and phospho6-tau expression in the three different brain regions with Western blotting .No differences in tau expression were seen among any samples. Two bands of different sizes representing phospho-tau (52 and 75 kDa) were detected and calculated. Each phospho6-tau expression was nominally increased (but not significant) in AD and control mice in an age-dependent manner. However, phospho6-tau (75 kDa) in the 6-month-old AD mice tended to be lower than that in the 3-month-old AD mice.To clarify the relationship between cognitive dysfunction in AD mice and neuronal function, we assessed the expression of neurotrophic factors and their receptor proteins . HoweverWe isolated mitochondria and measured protein expression using Western blotting. First, we checked the quality of our mitochondria isolation technique using our self-made buffer A. After To clarify the relationship between senescence and cognitive function in AD transgenic mice, we assessed their cognitive function using the Morris water maze task at three different ages. The goal time gradually decreased for all mouse groups with or without AD. The goal time of the final trial day gradually decreased in an age-dependent manner in both mouse groups. These results showed that cognitive function was gradually impaired by aging with or without AD and are consistent with our previous study . The goaTo clarify that these differences were not simply due to motor dysfunction, we measured swimming speed. We found no significant differences among the mouse groups. Additionally, we measured coordination ability using the rota-rod test. The fall time gradually decreased in an age-dependent manner in mice with or without AD. The fall time for the 20-month-old control group was significantly faster than that of the 3-month-old group. These results, using two different apparatuses, showed that AD mice did not induce much motor dysfunction and coordination disorder. Even during normal breeding, we found no particular difference in gait or movement in the AD mice. We found that the cognitive function of the AD mice simply deteriorated at a younger age. However, the fall time in the rota-rod test for the AD mice tended to be nominally longer (but not significantly so) compared to that of the age-matched controls. The reason is likely due to the difference in body weight. The fall time in the rota-rod test tends to be body-weight-dependent. In our previous study, obese mice fell faster than age-matched controls, and a negative correlation between body weight and fall time was observed . In thishttps://www.jax.org/strain/008730, accessed on 17 December 2021). This strain overexpresses both human Aβ precursor protein with the Swedish , Florida (I716V), and London (V717I) Familial Alzheimer’s Disease (FAD) mutations and human presenilin-1 harboring two FAD mutations, M146L and L286V. JAX recommends that this strain be used as an Aβ1-42-induced neurodegeneration model. To elucidate which AD-related proteins affected cognitive dysfunction in the Morris water maze trial, we examined Aβ1-40, -42, tau, and phospho-tau protein expression using Western blotting analysis.In this experiment, we purchased AD transgenic mice, which are widely used ,30. BasiAβ1-40 expression in the AD mice was increased in all brain regions in an age-dependent manner. However, the rate of the increase in its expression in the AD mice, especially in the 20-month-old mice, was lower than that of Aβ1-42, and the control mice also gradually showed an increase in Aβ1-40 in every brain region in an age-dependent manner. Aβ1-42 expression in the AD mice also increased in an age-dependent manner, except in the hippocampus of the 20-month-old mice. Surprisingly, the increased rate of Aβ1-42 expression in the hippocampus was much higher than that of the other two brain regions in all AD mouse groups. Generally, the toxicity of Aβ1-42 is considered to be stronger than that of Aβ1-40 , and Aβ1We found no significant difference in tau expression in any of our samples. We calculated phospho-tau expression using two different molecular weight bands, and the expression of the 75-kDa band gradually increased in an age-dependent manner in both the AD and the control mice, except in the 6-month-old AD mice. The increased rate of 75-kDa phospho-tau expression was higher than that of the 52-kDa band, and hippocampal expression of the 75-kDa band was higher than that in the other two brain regions in both the AD and the control mice. These results indicate that cognitive impairment in AD mice is likely induced by Aβ1-42 aggregation in the hippocampus.Neurotrophic factors such as NGF and BDNF play an important role in the maintenance of higher brain function. The functions of neurotrophic factors include axonal elongation, neuronal survival, and neurotransmitter synthesis . To clarROS attack many tissues and induce oxidative damage and cell death. The “free radical theory of aging” hypothesis was first proposed by Harman in 1956 . The accTo determine the mitochondrial oxidation levels, we measured the expression of proteins using isolated mitochondria. SOD-2 , which detoxifies superoxide, is specifically present in mitochondria. SOD-2 mutations are related to the onset of AD, Parkinson’s disease, and stroke . SOD-2 eNMNAT-3 levels were also attenuated in every region of AD mouse brains in this study. NMNAT-3 catalyzes the formation of nicotinamide adenine dinucleotide (NAD+) synthesis from nicotinamide mononucleotide and ATP, and is localized in the mitochondria . NAD+ isNQO-1 is a flavin-adenine-dinucleotide-dependent flavoprotein that serves as an oxidative stress response protein . NQO-1 eHowever, all analytical data for Western blotting of isolated mitochondria were normalized by COX-IV. COX-IV is one of the mitochondrial-specific proteins and is a subunit of cytochrome c oxidase in the mitochondrial respiratory chain. For Western blotting using isolated mitochondria, COX-IV, VDAC1/Porin, and heat shock protein 60 (HSP60) may be used as loading controls. However, care should be taken when using these proteins as loading controls. COX-IV may already be damaged in AD transgenic mice ,52. It iIn our experiments, we found that the AD mice showed significant damage from ROS. However, the number of samples of isolated mitochondria from AD mice was limited other than for the outside of the cerebral cortex region. If mitochondria can be protected from ROS, mitochondrial function and cognitive function may be maintained. Future studies should explore compounds that can specifically exert a strong antioxidant capacity in mitochondria.


Oxygen uptake in absolute (VO2) and relative to body weight (rel. VO2), lactate, and the rate of perceived exertion (RPE) were measured before and after the test. WB-EMS intensity was adjusted individually according to the feedback of the participant. The descriptive statistics were given in mean ± SD. For the statistical analyses, one-factorial ANOVA for repeated measures and two-factorial ANOVA  were performed (α = 0.05). Significant effects were found for EMS and W/NW factors for the outcome variables VO2 , relative VO2 , and lactate  and both the factors produced higher results. However, the difference in VO2 and relative VO2 is within the range of biological variability of ± 12%. The factor combination EMS*W/NW is statistically non-significant for all three variables. WB-EMS resulted in the higher RPE values , RPE differences for W/NW and EMS*W/NW were not significant. The current study results indicate that WB-EMS influences the parameters of exercise intensity. The impact on exercise intensity and the clinical relevance of WB-EMS-superimposed walking (WB-EMS-W) exercise is questionable because of the marginal differences in the outcome variables.Electrical muscle stimulation (EMS) is an increasingly popular training method and has become the focus of research in recent years. New EMS devices offer a wide range of mobile applications for whole-body EMS (WB-EMS) training, e.g., the intensification of dynamic low-intensity endurance exercises through WB-EMS. The present study aimed to determine the differences in exercise intensity between WB-EMS-superimposed and conventional walking (EMS-CW), and CON and WB-EMS-superimposed Nordic walking (WB-EMS-NW) during a treadmill test. Eleven participants (52.0 ± years; 85.9 ± 7.4 kg, 182 ± 6 cm, BMI 25.9 ± 2.2 kg/m Electrical muscle stimulation (EMS) is an increasingly popular training method and has become the focus of research in recent years. EMS is the electrical excitation and contraction of a muscle or a muscle group through an external applied electric field  training. Therefore, the use of superimposed WB-EMS for intensification of dynamic exercises has become more prominent in research  compared with level walking   and CON and WB-EMS-superimposed Nordic walking (WB-EMS-NW) during a treadmill test with a constant given velocity in a cohort of healthy men. We assume that WB-EMS-superimposed walking and Nordic walking (WB-EMS-W/NW) result in higher VOwww.randomizer.org).The study was conducted as a randomized crossover design with a total of four measurement appointments. To avoid WB-EMS-induced delayed onset muscle soreness (DOMS) as a confounding factor on test performance  to ensure their ability to perform the trial. During this examination, body weight (BW) and height were measured, and a physician performed a basic orthopedic and cardiopulmonary checkup which included a resting electrocardiogram. Blood samples were also taken to exclude acute infection.2) volunteered to participate. The ethics committee of the University of Potsdam approved the study (application number: 19/2018).The recruitment of participants took place in the environment of the research facility and a local sports center. Healthy, non-smoking men between the age of 35 and 70 years with a moderate activity profile (<3 h/week) were included. Regular drug consumption, cardiac arrhythmias (Lown classification ≥ 3), heart failure (New York Heart Association classification ≥ II), and wearing an implantable cardiac device were exclusion criteria. Additional exclusion criteria were diseases, such as chronic obstructive pulmonary disease (COPD), movement-restricting diseases, and rheumatic and neurological diseases. Eleven healthy men  with a low-frequency neuromuscular electrical stimulation with interval stimulation pattern  was used. For WM-EMS, the participants had to wear a trunk vest, a waist belt, and cuffs on/over the thighs and upper arms for the stimulation of the eight muscle groups . Due to the different electrical conductivity of the skin and tissues, WB-EMS intensity was adjusted individually and separately for each muscle group and the overall intensity during the WB-EMS test situations until the individual tolerated maximum amperage was reached. In contrast to previous studies that recommend a WB-EMS familiarization to achieve higher currents by the participants . Depending on the test situation, the participant was prepared with the WB-EMS equipment and if applicable NW sticks, which were adjusted in relation to body height (height sticks = body height × 0.68). Afterward, the treadmill test  took place . For assessment of the respiratory values, the wireless portable breath-by-breath system MetaMax 3B by Cortex was used . The MetaMax 3B system measures volume using a bidirectional digital turbine. Using the analysis software Metasoft version 3.9.5, the respiratory data, inspiration and expiration volume, and concentration of O2 and CO2 were calculated. The analyzing system was calibrated concerning volume, pressure, and gas  before every test. An air-conditioning system guaranteed stable conditions during the whole test in the lab (STPD). VO2 and relative VO2 were reported as an average value over the 10 min test.Outcome measures of interest were VOCapillary blood samples were taken from the earlobe before the test, in the 1 min break after warm-up and after finishing the test. Blood lactate concentration was determined with in-house used analysis devices . Subjective perception of participants (RPE) of exercise intensity was assessed using the Borg scale (6–20) before and after the test  with a test-retest variability within the range of biological day-to-day variability for VO2 measurements (± 12%) and good reliability of RPE measurements (ICC = 0.859).The reliability for VO*W/NW) were performed. Paired t-test was used to identify differences in WB-EMS current intensities to the individual WB-EMS current intensities of the participants as well as in the mean current intensity of the eight muscle groups between the two measurement conditions. For all tests, the statistical software SPSS 23.0 was used, a significance level of α = 0.05 was assumed.Results are presented descriptively (mean ± SD). The Kolmogorov–Smirnov test was used to test data for normality. Differences in outcome parameters between WB-EMS-W and CON-W to NW were presented relatively. BW differences between the four tests were analyzed using a one-factorial ANOVA test for repeated measures. To identify differences between WB-EMS-supported and CON-W respectively NW, two-factorial ANOVA with the single factors “EMS application” (EMS) and “(W/NW)” and its combination , 0.000 (W/NW), and 0.935 (EMS*W/NW). The temporal course of VO2 for the four test conditions is given in 2 with mean values from 20.11 ± 1.05 to 25.18 ± 1.83 ml/min/kg and p-values of p < 0.001 (EMS), p < 0.000 (W/NW), and 0.935 (EMS*W/NW).All subjects completed the measurements successfully and were included for data analyses. There were no dropouts and no missing data. The results of body weight, the four outcome measurements, and results of the statistical analyses are presented in p = 0.035). However, W/NW (p = 0.370) and the factor combination EMS*W/NW (p = 8.57) revealed no significant difference. The results for blood lactate concentration are in line with the results for VO2 and relative VO2 with a range of the mean values from 1.14 to 2.39 mmol−1, statistically significant results of the 2-factorial ANOVA for EMS (p = 0.003) and W/NW (p = 0.003), and a statistically non-significant result for EMS*W/NW (p = 0.117).Post-exercise RPE ranged from 11.09 to 12.64 with a statistically significant difference for EMS as an influencing factor . Muscle group WB-EMS current intensity ranged from 15.6 to 43.7 with a mean ± SD of 24.7 ± 8.4 for W and from 15.6 to 46.6 with a mean ± SD of 25.7 ± 3.8 for NW. The t-test revealed no significant difference (p = 0.054).Results of WB-EMS current intensity analysis are given in 2 uptake was higher in both EMS-W/NW when compared with CON exercises. In addition, WB-EMS leads to higher blood lactate concentrations when applied to exercise. However, clinical relevance for VO2 uptake and blood lactate concentration is debatable. The second finding of our study is that WB-EMS-superimposed exercises are perceived as more strenuous compared with CON exercise.The major finding of our study is that superimposed WB-EMS affect metabolic demand during W and NW significantly. VOTo our knowledge, this is the first investigation of a possible intensification of low-intensity exercises through superimposed WB-EMS. However, a wide range of studies already showed that EMS has the potential to increase the metabolic demands of different types of exercise, whether limited to a single muscle or related to the whole body  compared with the cycling group. Even though participants performed more demanding exercises for the metabolic and respiratory system with applied WB-EMS, a 4 week training intervention of WB-EMS-superimposed cycling exercise did not result in superior improvements of endurance capacity than cycling alone did. This fact is in line with our results that the increase of 9.41% (VO2) compared to 9.77% (relative VO2) for WB-EMS-W and 8.69% (VO2) compared to 8.2% (relative VO2) for WB-EMS-NW is within the range of biological day-to-day variability. Therefore, regardless of the fact that the differences we obtained are statistically significant , the intensification of W and NW through WB-EMS seems to be clinically irrelevant for this population. However, the results for combined factor analyses (EMS*W/NW) of the 2-factorial ANOVA test indicate that WB-EMS does not have a different impact on VO2 between WB-EMS-W/NW. This result contradicts our hypothesis that due to the higher muscle recruitment in various body segments during NW, WB-EMS has a higher impact on NW compared to W.Regarding WB-EMS-superimposed cycling, several studies support our findings regarding exercise intensification through WB-EMS. Wahl et al.  investigThis conclusion is also confirmed by the results of the EMS current intensity analysis. Various studies recommend a WB-EMS familiarization for a higher WB-EMS current intensity toleration by the participants and ultimately higher impact of the WB-EMS  and 14.09% (WB-EMS superimposed). NW has been the subject of research in previous studies, for example, Schiffer et al. . In contrast, Church et al. . In line with our results, they found an initial difference of ≈12.2% in VO2 between NW and W and beyond an increase of ≈17.6% after the learning period. Our participants were only fundamentally familiar with the NW technique. Therefore, it could not be excluded that a familiarization of the participants with the NW technique might have enlarged the difference in VO2 intake between W and NW. It can only be speculated about a stronger WB-EMS impact after an NW technique-learning period. However, a longer period of NW with additional WB-EMS might increase the VO2 uptake and relative VO2 uptake.An interesting result of our study is the difference in VOr et al.  investigh et al.  found a e et al.  investig2 uptake and RPE  in a group of chronic obstructive pulmonary disease patients and compared the results of two 6 min walking tests, one time during a standard W test and other time during NW. Participants did not report any differences in RPE between W and NW. Figard-Fabre et al.  and the occurrence of accompanying diseases (70 years), which could influence the outcome measures. However, the low SD in the examined variables suggests that age should not be regarded as an influencing factor. This could be because the selected exercise intensity for the W and NW, both conventionally and under WB-EMS, was a low-intensity exercise in a range in which the age and the associated VO2 capacity of the test persons have no influence.There are some limitations of the current study. The high variability in the age of the included participants within this sample can be stated as a limitation of the study with regard to the results, since it would be expected that there would also be a high scatter, especially with VOOur results show that the use of WB-EMS to intensify exercise is limited, at least in our subject group. An influence of the WB-EMS on exercise intensity is statistically shown but is below the threshold for clinical relevance. Therefore, the high cost of acquiring the device should be considered when using WB-EMS in different patient groups, such as T2DM and assessing training success. However, habituation of the subjects with the WB-EMS and the presumed higher tolerance to the current intensity of the WB-EMS and associated higher intensification may still change this cost-benefit consideration and indicate WB-EMS-W or NW as an effective training method in different patient groups such as T2DM.Our results indicate that WB-EMS influences parameters of exercise intensity. The impact on exercise intensity and the suitability for intensifying low-intensity exercise is questionable because of the low differences in the outcome variables. Regarding our results, CON-NW seems to be a more intensive training method than WB-EMS-W. Further studies seem to be necessary to confirm our results. An application of WB-EMS with more intensive ergometer exercises, such as elliptical machine training, could be another approach for WB-EMS-superimposed training. We acknowledge the support of the Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of University of Potsdam.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the University of Potsdam. The patients/participants provided their written informed consent to participate in this study.RV and JS conceived, designed the research, and analyzed the data. RV conducted the experiments and wrote the manuscript. JS, MH, AQ, and HV revised the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Antimicrobial stewardship (AS) is defined as coordinated interventions to improve and measure the appropriate use of antimicrobial agents. However, available resources for AS differ depending on the size of the clinical setting. Therefore, AS programs based on guidelines need to be selected in order to implement AS in small- to medium-sized hospitals. The present study compared the impact of AS in a 126-bed community hospital between pre- and post-AS periods.The present study was retrospectively performed by selecting data on eligible patients from electronic medical records stored in the central database of the hospital. The roles of the AS team included weekly rounds and recommendations on the appropriate use of antimicrobials, and pharmacists working on post-prescription audits and pharmaceutical care at the bedside closely communicated with the AS team to assist with its implementation. As process measurements, the order rate of culture examinations, the conducting rate of de-escalation, antimicrobial use density (AUD), days of therapy (DOT), and the AUD/DOT ratio of carbapenems and tazobactam-piperacillin (TAZ/PIPC) were measured. Thirty-day mortality and recurrence rates were examined as clinical outcomes.p < 0.01) from 10.4% in the pre-AS period to 21.1% in the post-AS period. The order rate of culture examinations increased from 56.3 to 73.3% (p < 0.01). The conducting rate of de-escalation increased from 10.2 to 30.8% (p < 0.05). The AUD of carbapenems and TAZ/PIPC significantly decreased (p < 0.05). The DOT of carbapenems (p < 0.01) and TAZ/PIPC (p < 0.05) also significantly decreased. The AUD/DOT ratio of carbapenem significantly increased from 0.37 to 0.60 (p < 0.01). Thirty-day mortality rates were 11.2 and 14.2%, respectively, and were not significantly different. The 30-day recurrence rate significantly decreased (p < 0.05) from 14.7 to 7.5%.A total of 535 patients (288 in the pre-AS period and 247 in the post-AS period) were enrolled in the present study. The recommendation rate to prescribers significantly increased (The implementation of AS in this hospital improved the appropriate use of antimicrobials without negatively affecting clinical outcomes. These results may be attributed to close communication between pharmacists working on post-prescription audits and pharmaceutical care at the bedside and the AS team. Antimicrobial resistance (AMR) increases the difficulties associated with treating an infectious disease and is a serious threat to human health. It has resulted in longer hospital stays, higher hospital costs, and greater mortality , 2. The AS is a medical stuff function that optimizes clinical outcomes while minimizing the unintended consequences of antimicrobial use, and includes the appropriate selection, dosing, route, and duration of antimicrobial therapy . RecommeTarumizu Chuo hospital is a 126-bed community hospital. The Infection Control Team (ICT) of the hospital include a physician (Infection Control Doctor: ICD), a clinical pharmacist without authorized infectious disease training, a nurse, and a clinical laboratory technologist, who concurrently have been members of AST since June 2019. Prior to the organization of AST, antimicrobial agents were administered at the discretion of the respective attending physicians. At that time, carbapenem or Tazobactam-Piperacillin (TAZ/PIPC) was administered to some cases as an empiric treatment for severe infectious diseases. Furthermore, antimicrobial agents were prescribed over long periods to some cases in which causative microorganisms were not identified. To improve these conditions, AST was organized in June 2019, and started weekly rounds and prospective audits of antimicrobial use with direct interactions and feedback. Regarding the prospective audit of antimicrobial use, pharmacists including an AST pharmacist working on post-prescription audits also started to check medical records in detail every day and also collected information on the conditions of patients as ward pharmacists. Pharmacists other than the AST pharmacist closely communicated with the AST pharmacist and supported the implementation of AS.Therefore, the present study was performed to assess the impact of AS on the use of antimicrobials and clinical outcomes at this 126-bed community hospital with close communication between pharmacists working on post-prescription audit, ward pharmacists, and AST.Staphylococcus aureus (MRSA), extended-spectrum β-lactamase-producing enterobacteria (ESBL-producing enterobacteria), carbapenem-resistant Enterobacteriaceae (CRE), vancomycin-resistant enterococci (VRE), multidrug-resistant Acinetobacter spp. (MDRA), multidrug-resistant Pseudomonas aeruginosa (MDRP). MDRA and MDRP were defined as strains that were resistant to carbapenems (minimum inhibitory concentration: MIC ≥16 mg/L), fluoroquinolones (MIC ≥4 mg/L), and amikacin (MIC ≥32 mg/L) based on the criteria for MDR strains specified by the Law Concerning the Prevention of Infections and Medical Care for Patients with Infections by the Japanese Ministry of Health and Welfare [The present study was conducted at Tarumizu Chuo Hospital, Tarumizu Municipal Medical Center. Study periods were the pre-AS period  and post-AS period . Eligible patients were inpatients treated with specific antimicrobials, administered parenteral antimicrobials for more than 14 days, with antimicrobial-resistant pathogens, and with a microbial-positive blood culture. Specific antimicrobials were meropenem (MEPM), imipenem/cilastatin (IPM/CS), TAZ/PIPC, levofloxacin, cefepime, cefozopran, vancomycin, and linezolid. Antimicrobial-resistant pathogens included methicillin-resistant  Welfare . PatientAS implementation in the pre-AS period included the registration system of specific antimicrobials when used, the monthly monitoring of antimicrobial consumption, the monthly monitoring of antimicrobial resistance, and annual lectures for hospital staff and occasional discussions with an attending physician. These activities were continued in the post-AS period. Regarding the post-prescription audit of antimicrobial use, dose adjustments based on the package inserts of medicines were implemented in the pre-AS period. Furthermore, careful checks of medical records, including prescriptions, laboratory test values, vital signs, and other conditions for eligible patients, were performed daily by pharmacists in the post-AS period. Pharmacists other than the AS pharmacist then closely communicated with the AST pharmacist and recommendations to prescribers from AST were implemented. Details on recommendations by AST to prescribers are shown in Table The present study was retrospectively performed by selecting data on eligible patients from electronic medical records stored in the central database of the hospital.Regarding process measurements of the appropriate use of antimicrobials, the order rate of culture examinations to identify pathogens and the conducting rate of de-escalation were examined and expressed as follows:We also examined AUD, DOT, and the AUD/DOT ratio of carbapenems (MEPM and IPM/CS) and TAZ/PIPC, which were more frequently used among the specific antimicrobials at this hospital. AUD and DOT were expressed as follows:The defined daily dose (DDD) was the value defined by the World Health Organization.We examined 30-day mortality and recurrence rates as the clinical outcomes of AS. The 30-day mortality rate was calculated as the number of patients who died within 30 days of the initiation of antimicrobial treatment divided by all participating patients. The 30-day recurrence rate was calculated as the number of patients with recurrence of the same disease within 30 days of discontinuing antimicrobial therapy divided by all participating patients.p-values of < 0.05 was considered to be significant.Statistical analyses were performed using ystat2018.xls for Windows . Nominal variables of the two groups were compared using the Chi-squared test and continuous variables using the Mann-Whitney U-test. A The present study was approved by the Ethics Committee of Tarumizu Chuo Hospital (No. 20–1). The Ethics Committee waived the need for informed consent since this study used anonymous, aggregate, retrospective data.A total of 535 patients were enrolled in the present study. The pre-AS and post-AS groups included 288 and 247 patients, respectively. The characteristics of patients enrolled in the present study are shown in Table p < 0.01). The number of cases recommended were 30 and 68, respectively. In the post-AS period, some patients received two or more recommendations. The acceptance rate of recommendations by prescribers was 93.3% (28/30) in the pre-AS period and 94.1% (64/68) in the post-AS period, which was not significantly different. The number of recommendations on the selection of antimicrobials increased the most in the post-AS period. The numbers of recommendations for de-escalation (7) and the duration of antimicrobial therapy (4) in the post-AS period increased from 0 cases in the pre-AS period.Details on recommendations by the AST for prescribers are shown in Table p < 0.01) from 56.3% in the pre-AS period to 73.3% in the post-AS period. The conducting rate of de-escalation significantly increased (p < 0.05) from 10.2% in the pre-AS period to 30.8% in the post-AS period.The order rate of culture examinations for the identification of microbial pathogens and the conducting rate of de-escalation are shown in Fig. p < 0.05) from 8.28 ± 3.39 in the pre-AS period to 3.67 ± 1.73 in the post-AS period. The AUD of TAZ/PIPC also significantly decreased (p < 0.05) from 15.05 ± 5.91 to 8.55 ± 3.47. The DOT of carbapenems significantly decreased (p < 0.01) from 21.62 ± 7.23 in the pre-AS period to 5.88 ± 1.76 in the post-AS period. The DOT of TAZ/PIPC also significantly decreased (p < 0.05) from 20.93 ± 8.76 to 12.65 ± 4.96. The AUD/DOT ratio of carbapenem significantly increased (p < 0.01) from 0.37 ± 0.07 in the pre-AS period to 0.60 ± 0.12 in the post-AS period, while that of TAZ/PIPC decreased from 0.73 ± 0.08 to 0.67 ± 0.07, which was not significant.AUD, DOT, and the AUD/DOT ratio of carbapenems (MEPM and IPM/CS) and TAZ/PIPC in both periods are shown in Table p < 0.05) lower in the post-AS period than that in the pre-AS period.Thirty-day mortality and recurrence rates in the pre- and post-AS periods are shown in Fig. As described above, a decreases were observed in the AUD and DOT of carbapenem and TAZ/PIPC in the present study. Based on these results, improvements in the appropriate use of antimicrobials were achieved without negatively affecting clinical outcomes by AS in this hospital.Small- to medium-sized hospitals have finite medical resources. Therefore, the implementation of ASPs has to be performed by selecting items available, and their impact may be limited. An assessment of the impact of implementing ASPs is important. Mas-Morey et al. evaluated 28 studies implemented in 26 hospitals with < 500 beds and reported that interventions were not generally associated with significant changes in mortality or readmission rates; however, substantial cost savings were achieved, mainly due to the reduced use of antibiotics or the administration of cheaper antibiotics . The preThe strategy of this study for ASP was to implement a prospective audit and feedback by AST closely communicating with ward pharmacists and pharmacists working on post-prescription audits. Dallit et al. highlighted 2 important core strategies for improving the impact of implementing ASPs: 1) a prospective audit with an intervention and feedback and 2) formulary restriction with preauthorization . AndersoThe present results showed that the recommendation rate to prescribers from AST significantly increased from 10.4 to 21.1% Table , and theThe conducting rate of de-escalation significantly increased from 10.2% in the pre-AS period to 30.8% in the post-AS period (Fig. Decreases were observed in the AUD and DOT of carbapenem and TAZ/PIPC, respectively Table , which iThe AUD/DOT ratio of carbapenem significantly increased Table . CarbapeIn the present study, a significant decrease was observed in the 30-day recurrence rate; however, in a review of 28 studies, Mas-Morey et al. reported no significant reductions in readmission rates after the implementation of ASP . The medThe present study had the following limitation: the relationship between clinical outcomes and AS was not clarified. Therefore, further progress in AS activity is necessary to improve clinical outcomes.The implementation of AS at this 126-bed community hospital significantly increased recommendations on the appropriate use of antimicrobials from AST to prescribers. It also increased the order rate of culture examinations and the conducting rate of de-escalation. Furthermore, decreases were observed in the amount and periods of use of carbapenems and TAZ/PIPC without a negative impact on clinical outcomes, such as 30-day mortality and recurrence rates, in the post-AS period from the pre-AS period. The beneficial effects of AS are considered to be achieved by close communication between pharmacists working on post-prescription audits and pharmaceutical care at the bedside and AST.


Ficus carica L.) from three cultivars, Taamriwthe (TH), Azegzaw (AZ), and Averkane (AV), at three ripening stages . Analyses was performed using Headspace Solid-phase Microextraction and gas chromatography coupled with mass spectrometry. Results revealed the presence of 29 compounds that were grouped into different chemical classes. Aldehydes comprised the most abundant VOCs identified in all the studied figs, while alcohols, ketones, and terpenes comprised the minor compounds found in TH, AZ, and AV figs, respectively. Different aroma descriptors were identified throughout the ripening stages of figs; fruity and green aromas were dominant in all cultivars, while a fatty aroma scarcely occurred in figs. A gallery plot representation demonstrated that certain VOCs differentiate the studied cultivars and the different ripening stages of figs. Principal component analysis findings demonstrated characteristic VOCs of distinct ripening stages and cultivars, those VOCs can be used as fingerprints to distinguish different cultivars and/or ripening stages.Aroma is one of the essential parameters that determine fruit quality. It is also an important feature of varietal characterization and so valuable for agro-biodiversity identification and preservation. In order to characterize changes in the aroma fingerprint through fig development, the main objective of the present research was to study the volatile organic compound (VOC) profiles of figs ( Ficus carica L.), which have always been appreciated for their sweet taste and high nutritional value, are an essential constituent of the Mediterranean diet alongside olive oil, cereals, and vegetables. The global fig production is around 1 million tons per year, with Turkey being the largest producer, followed by Egypt, Morocco, and Algeria  are involved in a wide range of functions. For example, VOCs contribute to fruit aroma and to protection against herbivores, microbial growth, and abiotic stress  was chosen for its high sensitivity and extraction efficiency , Azegzaw (AZ), and Averkane (AV). The three ripening stages  were distinguished by visual maturity characteristics based on color change and fruit firmness .For TH figs, the unripe stage (TH1) was identified by its yellow color and the firmness of the fruit, whereas the ripened fruit (TH2) tended to be softer and yellow on the surface. Fully ripe (TH3) figs tended to lose their firmness and the surface cracked slightly. For AZ figs, unripe (AZ1) fruit were always firm with green-yellow skin, while ripe ones (AZ2) were soft with a dark-green color. Fully ripe figs (AZ3) became softer with cracking skin. For AV figs, unripe fruit (AV1) were identified as having purple-green skin and being hard; as they began to ripen (AV2) they slightly lost their firmness and became purple. When these figs reached the fully ripe stage (AV3), the fruit turned dark and lost its firmness.Fig fruits were selected according to their color, size, and absence of physical damage. Then they were frozen (−20°C). Samples were homogenized with a blender (IKA A11 Basic) before analyses.Several methods can be used for sample comparison by SPME, including in-fiber standardization and traditional calibration methods using external and internal standards  stationary phase 50/30 μm, 1 cm length fiber was used for SPME . This fiber was chosen because of its efficiency in extracting a wide range of VOCs according to their molecular weight (40–275 g/mol) and polarity  was added with agitation (250 rpm) in the same conditions , in the same chromatographic conditions. Retention indices were determined according to the equation described by Babushok et al.  data library. Further identification was realized by calculating the retention indices (RIs) using a standard mixture of k et al. . The calP < 0.05. Principal components analysis (PCA) was analyzed using XLSTAT 5.01. A spider chart, drawn with Microsoft Office Excel 2013 , was used to illustrate aroma profiles.Differences in the evolution of fig volatile profiles during the three ripening stages were assessed by analyses of variance with one factor (ANOVA), performed using STATISTICA Software 7.1 . Mean values were analyzed by LSD (least significant difference) at a significant level of As shown in The results showed qualitative differences between the different chemical classes depending on the fig cultivars and ripening stages. Aldehydes were the most abundant compounds in TH figs while large quantities of both aldehydes and terpenes were present in AZ figs. Aldehydes were distinctive compounds of AV figs. Moreover, significant differences in the proportion of chemical classes in the same cultivar, depending on fig ripening state, were also noted.(P < 0.05). Six aldehydes were identified in cultivar TH, of which (E)-2-methylbut-2-enal contributes to the strong green fruit notes detected (P < 0.05) during the ripening process. During the development of TH figs the level of (E)-hex-2-enal increased 3-fold compared to the early ripening stage. Almost the same aldehydes were found in cultivar AZ with the exception of heptanal and (E)-oct-2-enal. In the case of the AV cultivar, only four aldehydes were extracted and a high content of (E)-hex-2-enal (31%) was detected.Aldehydes were the most abundant VOCs, demonstrated by their proportion of 50 ± 0.59%, 40.26 ± 0.27%, and 29 ± 1.20% of total VOCs for TH, AZ, and AV figs, respectively. Both TH and AV figs showed a significant increase in aldehyde content during the three ripening stages , whereasdetected . This alP < 0.05) in hexanal during the studied stages. Acetaldehyde was identified in all fig cultivars and demonstrated a significant (P < 0.05) 2-fold increase from the beginning to the final stage of ripening.Hexanal and benzaldehyde were the most abundant aldehydes in TH and AZ figs. All figs showed a significant decrease (P < 0.05) from the unripe to the ripe stage, when it was 76% higher. Limonene, epoxylinalol, and linalool were identified in cultivar AZ; it was observed that linalool was the most abundant terpene in AZ figs (87 ± 9.50%) and increased significantly (P < 0.05) throughout the ripening process. Only two terpenes were extracted from AV figs . 1,8-cineole has an herbal eucalyptus odor with an odor threshold that varies from 1.3 to 12 μg/kg  in esters was recorded during AV fig ripening. The TH and AV cultivars had higher total esters content, with proportions of 19 ± 0.48% and 25 ± 2.34%, respectively, whereas AZ figs contained lower proportions of esters (around 3%). The esters identified differed between fig cultivars. Four esters were detected in cultivar TH; ethyl acetate was the most abundant with 76 ± 10.60% of total esters and was present at a constant proportion from the unripe to the fully ripe stage. Ethyl 2-methylbutanoate and ethyl hexanoate were only detected in TH figs and made an essential contribution to the fruity aroma (P < 0.05) during the ripening of AV figs, leading to the perception of a fruity aroma with high odor thresholds  (A significant increase (ty aroma . AV figs0 μg/kg) .P < 0.05) in the alcohols produced by the three cultivars. Alcohol content significantly increased during the ripening of AV cultivars (There were significant differences (ultivars . Howeverultivars .E)-hex-2-en-1-ol were only detected in cultivar AZ, and were present during the unripe and ripe stages, respectively.Three different alcohols were identified in TH figs: pent-1-en-3-ol, 3-methylbutan-1-ol, and 2-ethylhexan-1-ol. The most abundant compound was 3-methylbutan-1-ol, which increased during fruit ripening. Four alcohols were extracted from the AV cultivar, among these pentan-1-ol was only identified at the ripe stage . In the P < 0.05). On the other hand, heptan-2-one significantly increased (P < 0.05) in both cultivars.Three ketones were identified in this study and two of them were found in all fig cultivars, with significant differences in occurrence. During the ripening period acetone significantly increased in TH figs and decreased in AV figs . The PCA results showed that the two first axes explained a total of 91.24 % (F1: 63.49% and F2: 27.75%) and 96.59% (F1: 89.66% and F2: 6.93%) of the total variance , which produced only a few results when compared to the SPME technique. Twenty VOCs were identified in both TH and AZ figs, it is notable that the TH fig is a dominant cultivar in Algerian orchards because it is highly valued in both fresh and dried form. Only 14 compounds were found in AV figs, highlighting the difference in the VOC profile of the different cultivars.Several research already focused on the study of the volatile profile of fresh figs, dried figs, fig liquors, and fig spirits -hex-2-enal were the most important aldehyde molecules detected; these are important aroma compounds in figs, contributing to their fresh green odor and providing a fruity flavor due to their low odor thresholds -hex-2-enal is biosynthesized from linolenic acid through the lipoxygenase pathway -hex-2-enal production is augmented due to the activity of lipoxygenase on membrane lipids during fruit ripening -hex-2-enal observed in the present study in fully ripe TH and AV figs. Similar results have been observed by Song et al. . The second is the methylerythritol phosphate pathway which contributes to the production of hemiterpenes (C5), monoterpenes (C10), and diterpenes (C20)  in AZ (changing from 37.40 ± 1.37 to 37.06 ± 2.56%) and AV figs (changing from 41.11 ± 4.43 to 58.89 ± 10.40%) in AZ figs, which were characterized by high terpene contents. The major terpene molecule was linalool, which is recognized for its floral scents and has several biological properties   and AZ (37.40 ± 1.37%), these molecules were produced in higher proportions in AV figs (58 ± 1.4%) which impacted AV figs aroma notes. Indeed, AV figs are characterized by aromatic notes provided by terpenes in comparison to the other cultivars. Terpenes are well-known for their contribution to pleasant sensory notes, as they are characterized by herbaceous, fruity, citrus, and floral scents  are biosynthesized from unsaturated fatty acids which are oxygenated by the lipoxygenase enzyme  in most samples, but reached 75.93 ± 4.40% in fully ripe TH figs. The finding of small quantities of ketones in fig aroma is similar to previous studies of fresh figs by Gozlekci et al.  and OlivE)-2-methybut-2-enal and β-caryophyllene, which are synthesized in considerable quantities by TH figs, as well as benzaldehyde and linalool, which are released at high levels by the AZ cultivar -oct-2-enal while the ripe AZ fig was distinguished by only one alcohol [(E)-hex-2-en-1-ol]. Moreover, the two earlier ripening stages of AV figs were characterized by 1,8 cineol (AV1) and pentan-1-ol (AV2).Some volatile compounds were particularly identified at different ripening stages of each cultivar; these VOCs could be considered to represent fingerprints of the ripening stages. Among these compounds, α-santalene was exclusively identified in fully ripe TH figs, and ethyl 2-methylbutanoate and ethyl hexanoate were the two esters that characterized the fully ripe state of TH figs. In addition, the unripe AZ cultivar was distinguished by the presence of benzyl alcohol, heptanal, and (E)-hex-2-enal, ethyl acetate, and acetone. From examination of E)-hex-2-enal.As shown in The increasing trend seen in VOC chemical classes during ripening may be due to the climacteric nature of figs. Indeed, Lalel et al.  establisThe VOC analyses performed on the three fig cultivars at three ripening stages revealed the presence of a total of 29 compounds belonging to five chemical classes. Overall, 20 VOCs were identified in both TH and AZ figs, showing that there is no difference between the total VOC content of a dominant cultivar (TH) and one which is endangered (AZ). However, only 14 compounds were found in AV figs. Fig aromas are represented by different aromatic descriptors including high fruity, green notes and a moderate sweet and floral aroma, as well as a slight note of fatty aroma. In this investigation, we demonstrated that cultivars and/or ripening stages can be distinguished by VOCs that can be considered as specific fingerprints and are also of interest for identification in examining the agro-biodiversity of figs. Of course, further analyses is required to study the genetic and environmental factor that can affect the production of VOCs. The study of the bound aroma form of VOCs during the ripening process will be interesting of itself and help researchers to better understand the natural dynamic process of fig aroma biosynthesis.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.KZ, DEK, and M-LF conceptualized the research. KZ and MB-b contributed to the data creation and performance of the software. KZ wrote the original draft and conducted the formal analyses. DEK and M-LF contributed to the methodology, supervision, and validation of work. All authors reviewed and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The average recall/sensitivity and specificity of IntSplice2 by fivefold cross-validation (CV) of the training dataset were 0.764 and 0.884, respectively. The recall/sensitivity of IntSplice2 was lower than the average recall/sensitivity of 0.800 of IntSplice that we previously made with support vector machine (SVM) modeling for the same intronic positions. In contrast, the specificity of IntSplice2 was higher than the average specificity of 0.849 of IntSplice. For benchmarking (BM) of IntSplice2 with IntSplice, we made a test dataset that was not used to train IntSplice. After excluding the test dataset from the training dataset, we generated IntSplice2-BM and compared it with IntSplice using the test dataset. IntSplice2-BM was superior to IntSplice in all of the seven statistical measures of accuracy, precision, recall/sensitivity, specificity, F1 score, negative predictive value (NPV), and matthews correlation coefficient (MCC). We made the IntSplice2 web service at https://www.med.nagoya-u.ac.jp/neurogenetics/IntSplice2.Prediction of the effect of a single-nucleotide variant (SNV) in an intronic region on aberrant pre-mRNA splicing is challenging except for an SNV affecting the canonical GU/AG splice sites (ss). To predict pathogenicity of SNVs at intronic positions −50 (Int-50) to −3 (Int-3) close to the 3’ ss, we developed light gradient boosting machine (LightGBM)-based IntSplice2 models using pathogenic SNVs in the human gene mutation database (HGMD) and ClinVar and common SNVs in dbSNP with 0.01 ≤ minor allelic frequency (MAF) < 0.50. The LightGBM models were generated using features representing splicing  RNA splicing is an essential process to generate mature mRNAs from precursor mRNAs, especially in higher eukaryotes . RNA splThe major pipelines of our analysis are indicated in To make IntSplice2 models, we used essentially the same 110 features that were used to make IntSplice . BrieflyTo make IntSplice2 models using Training Dataset-1787, we first optimized hyperparameters with Optuna  and thenFor benchmarking (BM) the performance of LightGBM-based IntSplice2 against that of SVM-based IntSplice, we made Test Dataset-288 that was composed of 288 pathogenic and 288 common SNVs with 0.01 ≤ MAF < 0.50, which were not included in the Training Dataset for IntSplice . ExclusiIn an effort to make a new dependable model to predict the splicing effects of Int-SNVs at positions from Int-50 to Int-3, we made IntSplice2 using newly available SNVs and LightGBM modeling. We first asked whether the inclusion or exclusion of major SNVs that are observed in more than 50% in humans would improve the performance of generated models. We compared IntSplice2 models generated using common SNVs with 0.01 ≤ MAF < 0.50 and with 0.01 ≤ MAF < 0.99 by fivefold CV. SNVs with MAF > 0.50 indicate that the reference nucleotide represents a minor nucleotide. We found that common SNVs with 0.01 ≤ MAF < 0.50 gave rise to better scores in seven out of nine statistical measures than those with 0.01 ≤ MAF < 0.99 . We thusTraining Dataset-1787 was divided into Test Dataset-288, which was not used to train IntSplice, and Training Dataset-1499. We generated the IntSplice2-BM model using Training Dataset-1499. The average recall/sensitivity and the average specificity of IntSplice2-BM by fivefold CV were 0.764 and 0.889, respectively. We next compared the performances of IntSplice2-BM and IntSplice models using Test Dataset-288 and found that all the seven statistical measures were higher with the IntSplice2-BM model than the IntSplice model .We next compared LightGBM with random forest (RF), SVM, extremely randomized trees (ERT), and multilayer perceptron (MLP). The best hyperparameters of RF, SVM, ERT, and MLP were searched for by fivefold CV grid search, and the models were generated using scikit-learn libraries  on Pythohttps://www.med.nagoya-u.ac.jp/neurogenetics/IntSplice2. The IntSplice2 web service accepts a genomic coordinate according to either GRCh37/hg19 or GRCh38/hg38. A given coordinate is mapped to all the annotated coding transcripts in Ensembl release 101, and the web service program analyses all the transcripts. The program automatically generates three possible SNVs at the coordinate and predicts a probability of aberrant splicing, where 0 indicates that the SNV should have no effect on splicing and 1 indicates that the SNV should affect splicing of the downstream exon. A probability of aberrant splicing less than 0.5 was predicted to be a splicing-insensitive SNV, whereas that of 0.5 or more was predicted to be a splicing-affecting SNV. When an SNV is located at Int-50 to Int-3 of two or more transcripts, the web service program predicts the effects on splicing for all the relevant transcripts. Representative results are shown in CDH23 and activates a cryptic splice acceptor site with the insertion of seven intronic nucleotides (We generated a web service of IntSplice2 at leotides .In this study, we generated IntSplice2 using an updated dataset of pathogenic and common SNVs with LightGBM modeling. In contrast to IntSplice2, our previous tool IntSplice used SVM modeling . We compcis-elements as features. RegSNPs-intron additionally used the protein structure and the evolutionary conservation as features. In contrast to these models, MMSplice was a DNN-based model that was trained by true donor and acceptor sites to predict the effects of genetic variants on splicing (Recently, four prediction tools were developed using ML approaches for Int-SNVs : RF-basesplicing . We compsplicing . ML toolsplicing . In contThe original contributions presented in the study are included in the article/in silico analyses. J-iT, SF, and KO wrote the manuscript. All authors read and approved the final manuscript.KO conceived the idea. J-iT, SF, AT, and AS designed the methods and performed The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Blood samples were taken at baseline, immediately after exercise, and after 20 min of recovery. The IL-6, IL-10, and TNF-α levels and the AAT activity increased significantly immediately after exercise. The IL-6 level was significantly higher after CWI-REST than after RT-REST. No changes in the activities of the lysosomal enzymes were observed. The effect of a 3 min CWI on the level of inflammatory markers during post-exercise recovery was limited. Thus, it might be considered as a widely available method of regeneration for recreational athletes.Cold-water immersion (CWI) after exercise is a method used by sportsmen to improve recovery. The aim of the study was to assess the effect of a 3 min CWI on the inflammatory state by measuring levels of interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor α (TNF-α), and transforming growth factor β1 (TGF-β1), and activities of α1-antitrypsin (AAT) and lysosomal enzymes, including arylsulfatase (ASA), acid phosphatase (AcP), and cathepsin D (CTS D), in the blood of healthy recreational athletes. Male volunteers ( Physical exercise is known to enhance and maintain overall health and wellness . HoweverMany athletes are interested in regeneration methods based on the exposure of the body to low ambient temperatures, i.e., during a bath in cold water. A rapid increase of body temperature after the end of the low temperature exposure causes increases in the blood flow, leading to better removal of waste metabolites and inflammatory mediators released by damaged tissue . In addiTaking into account the unexplained issues of the impact of short CWI on inflammation, the aim of the present study was to determine if a 3 min CWI applied post-exercise is long enough to diminish the level of inflammatory markers in the blood of recreational athletes. For this purpose, concentrations of selected cytokines, including IL-6, IL-10, TNF-α, and transforming growth factor β1 (TGF-β1), as well as activities of AAT and selected lysosomal enzymes, including arylsulfatase (ASA), acid phosphatase (AcP), and cathepsin D (CTS D), were measured in the blood of young healthy recreational athletes who performed a 30 min submaximal aerobic exercise followed by a 20 min rest at room temperature with or without an initial 3 min cold-water bath.Twenty-two young healthy recreationally trained male athletes who participated in the summer camp voluntarily agreed to participate in the study. The research included men who had never used cold-water immersion before the study period. The inclusion and exclusion criteria are described in 2/min/kg and represents the baseline oxygen consumption [The International Physical Activity Questionnaire (IPAQ) was used to assess the level of physical activity of the subjects during the last 7 days before the study. The IPAQ is expressed in MET × min/week. One MET is equal to 3.5 mL Osumption . The incsumption . In the 2), body fat , and total body water mass , were determined using a Tanita bioelectric impedance analyzer—BC 418 MA . A physical working capacity-170 (PWC170) test was performed to determine the aerobic fitness of the participants [2 max), which is the primary oxygen function index [2 max variables of all participants were calculated according to the Astrand–Ryhming normogram, using the values of PWC170 test [The anthropometric evaluation and body composition of the participants, including body height , body weight , body mass index . Subsequently, serum was separated and stored at −80 °C for further analysis.The research project was divided into two sessions. The participants were assigned to either control condition (Session 1) or cold-water immersion (Session 2) in a counterbalanced crossover order. During Session 1, the volunteers were subjected to a 30 min exercise  on a bicycle ergometer (Monark Ergomedic 828 E), followed by a 20 min rest at room temperature in a sitting position (RT-REST). The HRmax was calculated according to Tanaka’s formula: HRmax = 208 − 0.7 × age . SessionThe activity of AAT was estimated by the Eriksson method . The basThe concentration of cytokines was estimated using ready-to-use immunoassays: IL-6 , IL-10 , TNF-α , and TGF-β1 . The measurements were made according to the manufacturer’s instructions. The cytokine concentrations were expressed in pg/mL or in ng/mL. The sensitivity of the methods, depending on the used calibrates, was 2 pg/mL for IL-6, 4.9 pg/mL for IL-10, 8.6 pg/mL for TGF-β1, and 8 pg/mL for TNF-α.All the studied parameters were analyzed in duplicate, with sample means taken as the result.p < 0.05 were accepted as statistically significant. The effect size (ES: Cohen’s d) was used to measure the difference between sessions, using the following formula: Cohen’s d = (M2 − M1)/SDpooled, where: SDpooled = √((SD1 + SD2)/2). ES magnitudes were interpreted as follows: <0.2 trivial; 0.2–0.6 small; 0.6–1.2 moderate; 1.2–2.0 large; 2.0–4.0 very large; 4.0 nearly perfect [The obtained results underwent univariate analysis of variances (ANOVA) with post hoc statistical analysis (Tukey’s HSD test and Tukey’s assay for different N). To calculate the sample size, the power of 80% and alpha level of 0.05 were used. In the post hoc analysis, all the assumptions of ANOVA  were considered. The results are presented as an arithmetic mean ± standard deviation (SD). Differences at the level of significance  perfect .2 max value points to the average aerobic fitness of the subjects [The characteristics of the studied group is presented in subjects . Using tsubjects . No incip < 0.001) and was still significantly higher (p < 0.001) after 20 min of rest at room temperature (RT-REST) than before exercise (BE)  (p < 0.05), and additionally increased (p < 0.05) after 20 min of rest with an initial 3 min cold-water immersion (RT-CWI)  (p < 0.05) and after 20 min of recovery (p < 0.001), when compared to the concentration of this cytokine BE. In Session 1, the concentration of IL-10 was about two-fold higher AE compared to its BE level (p < 0.05). Furthermore, the concentration of IL-10 after RT-REST was five-fold higher than AE (p < 0.001) and about ten-fold higher than BE (p < 0.001)  (p < 0.05). Moreover, the concentration of IL-10 after CWI-REST was five-fold higher than AE (p < 0.001) and about eleven-fold higher than BE (p < 0.001)  (p < 0.05). After RT-REST, the concentration of TNF-α statistically increased, as compared to the result obtained AE (p < 0.05)  (p < 0.05)   in Session 1 (ctively) a. In Sesctively) a. In botctively) b. A simictively) b. In Sesctively) c. A simictively) c. No stactively) d.p < 0.001) after CWI-REST than after RT-REST . The ES between session comparisons of the IL-10 concentration was moderate (0.87). The concentration of other cytokines did not differ in a statistically significant manner, comparing RT-REST and CWI-REST. For IL-6, TNF-a, and TGFβ1, the ESs were small .The results obtained in both sessions were compared. The concentration of IL-10 was significantly higher (p < 0.05). After RT-REST, AAT activity was lower than AE, but it remained higher than BE (p < 0.05)  (p < 0.05) , but decreased to the BE value after CWI-REST (0.72 ± 0.08 mg inhibited trypsin/mL)  a. In Sespsin/mL) a. No stapsin/mL) b–d.The activity of lysosomal enzymes and AAT did not differ in a statistically significant manner, comparing RT-REST and CWI-REST. For AAT, AcP, and ASA, the ESs were small  and for CTS D, the ES was large (1.44). The large effect size in the case of CTS D with no statistically significant differences between recovery with and without CWI may indicate that the sample size was not big enough.The aim of the present study was to determine if a 3 min CWI applied post-exercise is long enough to diminish the level of inflammatory markers in the blood of recreational athletes. Statistically significant increases in IL-6 and IL-10 concentrations were observed in the blood serum of the participants after submaximal exercise. However, a significant post-exercise decrease in TNF-α concentration was observed, followed by a statistically significant increase in the level of this cytokine after RT-REST. Moreover, no statistically significant changes in TGF-β1 concentration were observed. The finding of triggered cytokine production as an effect of physical exercise concurs with many previous studies ,27,28,29The post-exercise increase in IL-6 levels is well documented in existing literature ,33,34,35A decrease in TNF-α concentration immediately after exercise, observed in the present study, may indicate that submaximal exercise of the volunteers during the experiment did not cause any significant muscle cell damage. TNF-α concentration is supposed to depend on the secretion of IL-6 as this cytokine inhibits TNF-α expression . HoweverIn the present study, statistically significant post-exercise increases in the AAT activity were observed during both studied sessions. Markovitch et al.  demonstrAlthough many studies have investigated the cytokine response to exercise, only a few of them have validated the anti-inflammatory effects of CWI. A delayed change in the IL-6 level and a decrease in the TNFα level were reported after a 10 min CWI  and afteIn the present study, the ESs of comparisons between the two recovery methods used were small for most of the measured cytokines. In both sessions, the IL-6 level was statistically higher immediately after exercise. However, a further significant increase in the cytokine concentration was observed only after CWI-REST. This result is in line with the findings of Roberts et al. , who revRhind et al.  revealedIn the present study, it was also found that the estimated ES of AAT activity in comparisons between RT-REST and CWI-REST was small. Nevertheless, the AAT activity after CWI-REST was observed to return to the baseline value, while after RT-REST, the AAT activity remained statistically higher than before exercise. It may indicate the beneficial effect of short CWI on the risk of proteolytic tissue damage. The fast response of the AAT activity after different recovery methods, determined in the present study, may confirm its role as an acute phase protein in humans. The primary function of the acute phase response is to protect the organism from further injury and help restore homeostasis . AAT wasConsidering the activities of the lysosomal enzymes, no statistically significant changes were observed in both sessions of the present study. The estimated ESs for ASA and AcP activities were also small. This may indicate that the use of short CWI after physical exercise did not cause lysosomal membrane lability, as the lysosomal enzymes did not leak into the bloodstream. It was proven that lysosomal enzymes are involved in the development of post-exercise muscle fiber damage ,55. LysoThe limitations of the present study are the small number of the experiment participants and the relatively short duration of cold exposure. Future research should consider extending the cold-water immersion time and collecting blood samples at greater intervals after cold exposure.In conclusion, the results of the present study confirm that aerobic exercise induces the inflammatory response of the organism and add to the existing knowledge of the effects of cold-water immersion after exercise. It is worth emphasizing that the effect of CWI on the natural physiological stress and immune responses remains controversial. An effective protocol for using CWI as a method of post-exercise recovery has not yet been well established. The protocols used so far are largely based on individual experience rather than evidence-based research. Moreover, much research in this area is limited to the study of participants who were experienced (adapted) cold-water swimmers, professional athletes who exercised acutely in the cold, or athletes who were previously immersed in cold water as recovery. In the present experiment, the study group consisted of recreational athletes who had never been subjected to cold exposure before, but CWI limited to 3 min seemed harmless to them. The effect of short CWI on the level of inflammatory markers during post-exercise recovery appeared to be limited. Additionally, a slight beneficial effect of a 3 min CWI on the risk of proteolytic tissue damage was observed. Thus, short exposure to cold might be considered as a widely available and cheap method of recovery improvement in recreational athletes. However, the risk of exposure of the human organism to low temperature, in relation to the expected beneficial effects of recovery, as well as the most appropriate CWI protocols must be verified by further studies, in which the time of CWI or the time of blood collection will be extended.


Our hospital encountered the first coronavirus disease 19 (COVID-19) pneumonia death in Japan. Moreover, we prevented nosocomial infection by taking appropriate infection control measures, without a negative pressure chamber. The patient was an 82-year-old woman who had no history of traveling to Wuhan or any direct contact with individuals who had been to Wuhan. Our patient had a seven-day history of fatigue, sudden fever, and hypoxemia. Chest computerized tomography images revealed peripheral ground-glass opacities in her lungs. A diagnostic COVID-19 reverse-transcription polymerase chain reaction (RT-PCR) analysis was positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The patient did not respond to any treatment and died 13 days after admission. The possibility of COVID-19 in a patient must always be considered, especially in the current scenario, to prevent nosocomial infection from spreading. The initial cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia occurred in Wuhan, Hubei Province, China, in December 2019 . A varieAn 82-year-old woman was admitted to a hospital with a seven-day history of fatigue and sudden development of fever and hypoxemia. The patient’s body temperature (BT) was 38.9℃, and oxygen saturation was 92% (room air) at that time. Chest CT showed peripheral ground-glass opacities (GGOs), predominantly in both lower lobes of the lungs Figure . After fOn presentation , BT was 37.0℃ without the use of antipyretics, blood pressure was 148/77 mmHg, respiratory rate was 38 breaths/min, and pulse was 89 beats/min. Oxygen saturation was approximately 90% using 6 L/min oxygen supplementation via a reservoir bag. Laboratory tests showed elevated levels of white blood cells (WBCs) and C-reactive protein (CRP) Table .The patient had never smoked and did not have any relevant medical history except for the mediastinal tumor.Our patient neither had been to Wuhan nor had any direct contact with individuals that had been to or come back from Wuhan or were symptomatic with COVID-19. However, some individuals near the patient’s family were affected by pneumonia and showed signs of illness of unknown cause.Taking these into consideration, we suspected that the patient might be infected with SARS-CoV-2. Thus, when the patient was transferred to our hospital, we asked the health center if we could perform a diagnostic COVID-19 reverse-transcription polymerase chain reaction (RT-PCR) assay. However, the center declined because she did not meet the definite criteria. In Japan at that time, all the criteria from one of the following options had to be met for COVID-19 testing via polymerase chain reaction: (1) BT ≥ 37.5℃, respiratory symptoms, and traveled to or resided in Hubei within 14 days before the onset of symptoms; (2) BT ≥ 37.5℃, respiratory symptoms, and had been in close contact with those who had traveled to or lived in Hubei within 14 days before the onset of symptoms; and (3) fever or respiratory symptoms and had been in close contact with patients with COVID-19.Although we could not perform the diagnostic RT-PCR assay, an infectious disease, including COVID-19, was strongly suspected. Therefore, we admitted her to a private room as our hospital does not have a negative pressure room. All hospital staff that came in contact with the patient wore gloves, long-sleeved disposable gowns, protective eyewear, and surgical masks. In situations where the emission of aerosols was possible, the staff wore N95 masks.After admission to our hospital, we initiated noninvasive positive pressure ventilation (NPPV) (because the patient did not consent for intubation) and administered the antibiotics meropenem 1.5 g/day) and levofloxacin (500 mg/day) as well as the antiviral peramivir 300 mg/day), and corticosteroids . From the patient’s arterial blood gas results, we diagnosed acute respiratory distress syndrome (ARDS) and started sivelestat sodium hydrate. After starting these treatments, the patient’s subjective symptoms began to improve. Although her oxygen saturation and oxygen demand did not change, a chest x-ray showed an improvement in the shadow  who had been in contact with the patient were tested for COVID-19 via PCR analyses of pharyngeal swabs, and all results were negative. At that time, antibody tests for IgG and IgM had not been developed.We report the first case of death due to COVID-19 in Japan. Moreover, we could prevent nosocomial infection because the possibility of COVID-19 infection was considered from the beginning.Our patient had few symptoms. However, regarding the symptoms, 98.6% of patients have a fever, 44%-69.6% have fatigue, and 59.4%-76% have cough ,7. Even The source of infection in our patient is not certain. However, a follow-up investigation by the health center revealed that the pneumonia outbreak in the individuals near the patient’s family was caused by SARS-CoV-2. We suspect this could be the source of infection in our patient.In parallel with our case, COVID-19 patients unrelated to Wuhan nor had any contact with COVID-19 were detected in several areas of Japan. Thus, domestic community-acquired infection of COVID-19 was strongly suspected. A few days after our case, the criteria for COVID-19 testing had been relaxed, allowing testing for patients with BT ≥ 37.5℃, respiratory symptoms, and suspected pneumonia requiring hospitalization. This might have helped to clarify the infection situation in Japan.Nosocomial infections with SARS-COV-2 have been reported in several hospitals ,8, and iWe used NPPV in this case. NPPV is useful to support respiration, but it requires strict infection control because it can spread the virus to other individuals . In thisWe encountered the first death in Japan attributed to COVID-19 pneumonia using NPPV. In a hospital environment without a negative pressure chamber, following the appropriate infection control protocols can prevent nosocomial infections under the possibility of airborne transmission. We hope that the measures in this case contribute to the evidence of infection control against SARS-CoV-2.